Classical Methods for the Isolation of Nucleic Acids from Microorganisms
With the advent of molecular biology, an increasing number of diagnostic methods are based on the detection of nucleic acids. Nucleic acid amplification technologies represent useful tools in molecular biology. Since the discovery of the polymerase chain reaction (PCR), various protocols have been described for isolating nucleic acids suitable for detection and identification of microorganisms. However, most of these protocols are time-consuming and often require the use of toxic chemicals. In addition, protocols need to be tailor-made for each microbe type; a lysis protocol for fungi may not be suitable for gram-negative bacteria, or parasites, or bacterial spores, and so on. Furthermore, these protocols require numerous steps, increasing the risk of sample-to-sample or carry-over contamination.
Hugues et al. (Methods in Microbiology, 1971, vol. 5B, Academic Press, New York) have reviewed the available methods for disintegrating microbes for preparing biologically active fractions. The method selected will depend on its capability to process samples of a certain size or to be able to process multiple samples in a reasonable period of time, while the desired nucleic acids retain their integrity. Classical physical methods of cell breakage include mechanical cell disintegration (crushing and grinding, wet milling, ultrasonics, hydraulic shear, freeze pressure), liquid or hydrodynamic shear (French press, Chaikoff press, homogenizers, wet mills, vibration mills, filters, ultrasonic disintegration) and solid shear (grinding, Hugues press). Chemical methods of cell disintegration are mostly aimed at modifying the cell wall or cytoplasmic membrane, or both, so that the cells either become leaky or burst due to the effects of turgor pressure. Methods include osmosis, drying and extraction, autolysis, inhibition of cell wall synthesis, enzymic attack on cell walls, bacteriophages and other lytic factors, and ionizing radiation.
Several methods of cell disruption, with or without solid particles, and involving physical agitation to release nucleic acids have been described. Examples include a cell disrupter from Amoco (U.S. Pat. No. 5,464,773) and the FastPrep® apparatus from Qbiogene (U.S. Pat. No. 5,567,050). However, no examples of reduction into practice were provided in those patents. Other commercial devices that may be used to lyse cells using similar shaking-type bead mills include the Micro-Dismembrator II from B. Braun Biotech (Allentown, Pa., USA) and the Mini-BeadBeater from BioSpec (Bartlesville, Okla., USA). However, most methods described in the literature using this type of apparatus require additional nucleic acid purification steps after the lysis step (Miller et al., 1999, Appl. Env. Microbiol. 65: 4715-4724; Cornejo et al., 1998, Appl. Env. Microbiol. 64: 3099-3101). In addition, most protocols rely on the presence of lysogenic chemicals to enhance mechanical cell lysis. A major drawback of these methods is that the chemicals used often adversely affect subsequent molecular biology processes, such as the detergent sodium dodecyl sulfate (SDS) in PCR.
Significant progress has been made in the last few years by manufacturers to improve the simplicity of release and purification of nucleic acids from microorganisms present in clinical specimens. However, for laboratory personnel, it is not obvious to make a choice in the plethora of commercially available nucleic acids extraction kits. Those kits exhibit variable performances in the preparation of various test samples containing diverse target microorganisms for nucleic acids testing (NAT). These kits have different total nucleic acids recovery, number of manipulation steps and time requirements. Example 2 shows a comparison of 8 different commercially available DNA extraction kits with the method described in this invention. The conclusion was that with regards to speed, total recovery of DNA and number of manipulation steps, the sample preparation method described below was the best protocol.
A Novel Extraction Method that is Simple, Rapid, Universal and Versatil
The method described in this invention is a Rapid Universal Cell Lysis and Nucleic Acids Preparation (RUCLANAP) protocol. It requires approximately 10 to 15 min, in a simple, flexible and efficient protocol. The main sequence of events underlying this invention are an initial mechanical cell lysis using solid particles in the presence of a chelating agent, followed by a control of substances inhibitory to polymerases as used in amplification reactions, or inhibitory to other steps of NAT assays. Said inhibitory substances are present in the test sample and/or are released upon cell lysis into the lysate. For most applications, no further purification of nucleic acids is required. Such a control can be effected by diverse ways such as heating, adsorbing, freezing, removing or diluting the NAT inhibitors in the sample or the lysate. The advantages of this method are simplicity, rapidity, efficiency, universality (effective with all microbial species)and low cost. Prior to the present invention, heat was used to lyse cells (U.S. Pat. No. 5,376,527), agitation with particles was used to lyse cells (U.S. Pat. Nos. 5,567,050 and 4,295,613, and PCT Publication No. WO 02/10333), agitation with particles in organic solvents was used to lyse cells (U.S. Pat. No. 5,643,767), and agitation with particles was applied to already heat-lysed cells to provide access to nucleic acids (U.S. Pat. No. 5,942,425). Prior to the present invention, the particular sequence of events (agitation with particles in chelating buffer followed by heat inactivation of PCR inhibitors) was applied to prepare DNA for molecular biology. However, in these earlier methods, cell lysis did not rely upon mechanical forces, but mainly on beat provided by a boiling step. For example, others have used the combination of a vortexing step in CHELEX®, a weak ion-exchange matrix, followed by boiling of the sample (Kessler et al., 1997, J. Chin. Microbiol. 35: 1592-1594; Drake et al., 1996, Food Res. Int. 29: 451-455; Yoshimi et al., 1993, Acta Pathol. Jap. 43: 790-791). In all cases, the very short vortex step was performed only to mix the CHELEX® ion-exchange matrix with the sample in order to sequester divalent cations, and bind compounds which inhibit PCR (Singer-Sam et al., 1989, Amplifications 3:11); while cell lysis was obtained during the heating step, a process that is not universal for all microbes, especially bacterial spores and yeasts cells. As revealed in this invention in Example 19, the heating steps during the PCR protocol are not sufficient to lyse Mycobacterium smegmatis. 
Patent publication WO 99/15621 describes a method for mechanically lysing bacteria or yeasts wherein a liquid sample comprising the bacteria or yeasts is placed in a container with particles having 90-150 μm, namely 100 μm for bacteria and the large ones having 400-600 μm, namely 500 μm for yeasts. This method is not optimal, since after 8 minutes of vortex, about 60% of S. epidermidis and C. albicans are lysed.
Patent publication WO 00/05338 discloses a <<magnetic>> vortex method for lysing cells. At least two sizes of particles are used to mechanically break the cells. The larger particles are composed of a material which respond to a magnetic field (iron, namely), while the smaller ones are non-magnetic beads. The large beads are placed in movement by actuating a magnetic device and the sample is crushed between the large and the small beads, the smaller being moved by the larger ones. The ratio of size between the small beads and the target cells is rather small (50/1) while the ratio of size between the large and small beads is relatively large (40/1). The large beads have therefore the function of crushing the target cells between them and the small beads to free the cell contents into the lysate solution. Further, this application only provides a <<qualitative>> appreciation of the lysis rate and of the integrity of the nucleic acids obtained therefrom. Only electrophoretic gels show the results of the process. Amplification process further performed on nucleic acids are very sensitive processes which require a high level of lysis liability and reproducibility. WO 00/05338 is totally silent on achieving this high standard with the disclosed <<magnetic>> vortex method.
Patent publication WO 02/10333 describes a method which may be adapted for different techniques: sonication, mechanical vortex and magnetic vortex. The method comprises at least three parameters selected from: a) 50-100% of particles mass with regards to the total mass of the sample to be treated, b) a ratio of particles of a small diameter to particles of a large diameter (when two sizes of particles are used) which is less than or equal to 50%, c) a lysis duration of 9 to 20 minutes, d) glass beads that are involved in the motion of the particles and not in the lysis per se which are in a number of less than 7; and e) iron beads that have the same role as in d) but for a magnetic vortex, which are in a number of 5 to 15. Again the lysing particles have the size as in WO 99/15621 (the small ones having 90-150 μm, namely 100 μm for bacteria and the large ones having 400-600 μm, namely 500 μm for yeasts). Larger particle size ratios are favoured when a mixture of particles sizes is used (more than 50%), along with a relatively long lysis duration. Except for sonication, the other agitation techniques require large non-lysing particles which are present to put the small particles into motion. These big particles may render the bead matrix more voluminous and bumpy at the interface with the lysate, increasing the dead volume, and hence rendering the recovery by pipetting of the latter more difficult.
A publication by Jaffe et al. (J. Clin. Microbiol., 2000, 38: 3407-3412) discloses the use of 1.0 g of 100 μm glass beads and 0.25 g of CHELEX-100™ ion-exchange matrix in a volume of 0.5 ml of bacterial sample to mechanically break cells and release nucleic acids, further followed by a heating step. The process which is called “Bead beating with Chelex” however lacks sensitivity towards certain species, namely methicillin-resistant S. aureus (MRSA) even if the sensitivity is acceptable for other bacteria easier to break up, such as gram-negative species. There is no suggestion in this reference to vary the size of beads to improve the sensitivity and the versatility (“universality”) of the method for recovering efficaciously nucleic acids from any microorganism. Furthermore, this method has not been tested with clinical samples.
During the 99th ASM General Meeting held in Chicago in 1999, (Abstract C-481), the present inventors have disclosed the results of a comparative study conducted with the present method and kit (“then referred to as the IDI DNA extraction kit”) with eight other kits (see Example 2). No details were given on the protocol for lysing the cells and the components involved therein.
The RUCLANAP method is universal for microorganisms, as one can use it to extract nucleic acids from viruses, bacteria, bacterial spores, fungi, parasites or from other eukaryotic cells, including animal and human cells. The basic RUCLANAP protocol is versatile, as it can easily be adapted, depending on the type of clinical sample used, in order to dilute or concentrate the microorganisms present in the sample, and/or to extract crude nucleic acids, and/or to control (inactivate, adsorb, dilute or remove) sample-specific inhibitors of NAT assays (Wilson, Applied Env. Microbiol., 1997, 63:3741-3751). For genetic analysis of RNA, RNase inhibitors may be added to the sample before nucleic acid extraction with RUCLANAP; no additional purification of total RNA is necessary for subsequent amplification and detection, such as with RT-PCR (see Example 18).
The applications for this method are numerous: the samples from which microbial nucleic acids have been successfully prepared using this method include microbial broth cultures, positive blood cultures, suspensions of microbial colonies grown on agar media, as well as a variety of biological samples including blood, plasma, platelet concentrates, urine, cerebrospinal fluid, amniotic fluid, meconium, wound exudate, stools, nasal swabs, throat swabs, anal swabs, vaginal swabs, vaginal/anal swabs, rectal swabs, and bovine milk. The efficiency of Streptococcus agalactiae lysis by the RUCLANAP method was estimated to 99.99%, as determined by viable counts (see Example 5). For detailed sample preparation protocols with various specimens, see Examples 2 to 12, 15, and 17-20. The method is also amendable to scaling up or down, and automation.
One concern in the handling of biological specimens is safety. Heating of a clinical sample containing infectious agents is one of the preferred methods to render samples safer for handling. U.S. Pat. No. 5,376,527 describes a lysis-effective amount of heat that is sufficient to render difficult-to-lyse cells of Mycobacterium tuberculosis non-infectious. We have found that the combination of the cell lysis step and the heating step of the RUCLANAP method also renders most clinical samples safer for handling. A heating and/or freezing step may be performed prior to RUCLANAP to render test samples safer for handling.